The long-term goals of this proposal are to define the cellular and molecular determinants of heme homeostasis in human nutrition. Heme is a significant source of bioavailable iron for enterocytes where heme-iron is absorbed and for macrophages where heme-iron is recycled. In the human intestine, dietary heme is more easily absorbed than inorganic iron and is the source for the majority of body iron from a western diet. Moreover, over 70% of the total body iron is present as heme in hemoglobin. Iron from heme is recycled by phagocytosis of senescent red blood cells in the phagolysosome of macrophages. As of yet, the genes and pathways responsible for heme transport in the human intestine and macrophages of the reticuloendothelial system remain poorly defined. As a hydrophobic and cytotoxic cofactor, heme must be transported in a highly controlled manner through membranes via specific intra- and inter-cellular pathways. Prior to the last funding period, the identity of ny heme importer was unknown. We established that the roundworm Caenorhabditis elegans is an excellent animal model to identify heme transport pathways because it synthesizes a large number of hemoproteins with human homologs but does not synthesize heme de novo. By exploiting the heme auxotrophy of C. elegans in the previous grant cycle, we successfully identified the first eukaryotic heme importer/transporter, HRG1, and its corresponding human homolog, a discovery which provided the first preliminary framework for heme transport and trafficking in animals. HRG1 is essential for macrophage iron homeostasis and transports heme from the phagolysosome to the cytoplasm during phagocytosis of senescent RBCs - a process called erythrophagocytosis (Cell Metabolism 2013). Importantly, our studies reveal that a HRG1 variant in humans is defective in heme transport. The studies in this proposal are designed to elucidate the precise mechanisms of heme transport by HRG1 at the molecular, cellular, and organismal levels. We seek to test the hypothesis that HRG1 is the elusive human intestinal heme transporter and part of an essential heme trafficking network. We will utilize (a) a cell biological approach to establish HRG1 as the intestinal heme transporter; (b) a genetic approach to elucidate the molecular consequence of HRG1 variants in human iron metabolism disorders; and (c) a biochemical approach to uncover additional upstream and downstream interactors of HRG1 comprising a functional heme trafficking network. Our goal is to obtain a comprehensive understanding of the pathways which mediate heme transport in mammals that have, heretofore, remained poorly understood.